Transgenic technology is described for the production of animals that exhibit symptoms of human Alzheimer's disease through the expression of the Alzheimer's precursor protein or a modified version thereof.
Alzheimer's Disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia ("The early story of Alzheimer's Disease", edited by Bick K, Amaducci L, and Pepeu G. (Raven Press, New York 1987). It is the leading cause of dementia in elderly persons. AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibit personality changes, socially inappropriate actions and schizophrenia ("A guide to the understanding of Alzheimer's Disease and related disorders", edited by Jorm AF.; (New York University Press, New York 1987). AD is devastating for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration. The most common problems in the Alzheimer's patient are inability to dress unaided, restlessness by day, urinary incontinence and sleep disturbances. The family members report embarrassment, anxiety, depression, and a decreased social life.
The impact of AD on society and on the national economy is enormous. It is expected that the demented elderly population in the United States will increase by 41% by the year 2000. It is expensive for the health care systems that must provide institutional and ancillary care for the patients at an estimated annual cost of $40 billion (Jorm, 1987; Fisher, LM: New York Times, Aug. 23, 1989 D1 "Alzheimer's Disease", edited by Reisberg, B.; (The Free Press, New York & London 1983). These factors imply preventive action must be taken to decrease AD incidence by allocating resources into AD research.
At a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1,000 grams. At a microscopic level, the histopathological symptoms of AD include neurofibrillary tangles (NFT), neuritic plaques, and degeneration of neurons. AD patients exhibit degeneration of nerve cells in the frontal and temporal cortex of the cerebral cortex, pyramidal neurons of hippocampus, neurons in the medial, medial central, and cortical nuclei of the amygdala, noradrenergic neurons in the locus coeruleus, and the neurons in the basal forebrain cholinergic system. Loss of neurons in the cholinergic system leads to a consistent deficit in cholinergic presynaptic markers in AD (Reisberg, 1983; "Alzheimer's Disease and related disorders, research and development" edited by Kelly WE; (Charles C. Thomas, Springfield, Ill. 1984).
AD is associated with neuritic plaques measuring up to 200 .mu.m in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal constituents of neuritic plaques is amyloid, which is stained by congo red (Reisberg, 1983; Kelly, 1984). Amyloid plaques are extracellular, pink- or rust-colored in bright field, and birefringent in polarized light. The plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mechanisms of AD neuropathy. However, available evidence strongly indicates two distinct types of genetic predisposition for AD. First, molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate, et al. Nature 349:704-706 (1991); Murrell, J, et al. Science 254; 97-99, 1991; Chartier-Harlin, M-C, et al. Nature 353, 844-846 (1991)). Second, in certain other families with a clear genetic predisposition to AD, the mutation maps to chromosome 21 but is distinct from the APP locus (Tanzi, R. E., et al. Nature, 331;528-530 (1988)).
Amyloid plaques are abundantly present in AD patients and in Down's Syndrome individuals surviving to the age of 40. The plaques are also present in the normal aging brain, although at a lower number. These plaques are made up of the amyloid .beta. peptide (.beta. peptide) (Glenner and Wong, et al., Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is also the main protein constituent in cerebrovascular deposits and neurofibrillary tangles. The peptide is a filamentous material that is arranged in beta-pleated sheets and has a molecular weight of 4.2-4.5 kd. It is a hydrophobic peptide comprising 39-42 amino acids. The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang, et al., Nature 325:733-735 (1987); Goldgaber, et al., Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190-4194 (1987); Tanzi, et al., Nature 331:528-530 (1988) and genomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai, et al., Gene 87, 257-263 (1990). Three forms of APP cDNAs (APP695, APP751, and APP770) have been isolated, and arise from a single precursor RNA by alternate splicing. The gene spans more than 175 Kb with 18 exons (Yoshikai, et al., 1990). APP contains three extracellular domains, a transmembrane region and a cytoplasmic domain. The .beta. peptide consists of 28 amino acids just outside the membrane and 14 residues of the hydrophobic transmembrane domain. Thus, the .beta. peptide is a cleavage product of APP normally found in brain and other tissues such as heart, kidney and spleen. .beta. peptide deposits, however, are usually found only in the brain, although Joachim et al., Nature 341:226-228 (1989) have reported .beta. peptide deposits outside the brain in the skin, intestine, and subcutaneous tissues of most AD patients.
The larger alternate forms of APP (APP751, APP770) consist of all of APP695 plus one or two additional domains. APP751 consists of all of APP695 plus an additional 56 amino acids which has homology to the Kunitz family of serine protease inhibitors (KP1) (Tanzi et al., 1988; Weidemann, et al., Cell 57:115-126 (1989); Kitaguchi, et al., Nature 331:530-532 (1988); Tanzi et al., Nature 329, 156 (1987). APP770 contains APP751 and an additional 19 amino acid domain homologous to the neuron cell surface antigen OX-2 (Weidemann, et al., Cell 57:115-126 (1989); Kitaguchi et al., 1988). APP is posttranslationally modified by the removal of the leader sequence and by the addition of sulfate and sugar groups.
Van Broeckhaven, et al., Science 248:1120-1122 (1990) have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248:1124-1128 (1990). The mutation substituted a glutamine for glutamic acid at position 22 of the .beta. peptide (position 618 of APP695). In addition, certain families are genetically predisposed to Alzheimer's disease, a condition referred to as familial Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate, et al., (1991); Murrell et al., 1991; Chartier-Harlin et al., 1991). These mutations co-segregate with the disease within the families and are absent in families with late-onset AD.
There are no proven animal models to study AD, although aging nonhuman primates seem to develop amyloid plaques of .beta. peptide in brain parenchyma and in the walls of some meningeal and cortical vessels. Although aged primates and canines can serve as animal models, they are expensive to maintain and need lengthy study periods. There are no spontaneous animal mutations with sufficient similarities to AD to be useful as experimental models. Various models have been proposed in which some AD-like symptoms may be induced by electrolysis, transplantation of AD brain samples, aluminum chloride, kainic acid or choline analogs (Kisner, et al., Neurobiol Aging 7;287-292 (1986); Mistry, J. S., et al., J Med Chem 29;337-343 (1986)). Flood, et al. (Proc. Natl. Acad. Sci. 88:3363-3366 (1986), reported amnestic effects in mice of four synthetic peptides homologous to the B peptide. Because none of these share with AD either common symptoms, biochemistry or pathogenesis, they are not likely to yield much useful information on etiology or treatment.
Transgenic mice with the human APP promoter linked to E. coli .beta.-galactosidase (Wirak, D. O., et al., The EMBO J 10;289-296 (1991)) as well as transgenic mice expressing the human APP751 cDNA (Quon, D, et al. Nature 352, 239-241 (1991)) or subfragment of the cDNA including the .beta. peptide (Wirak, D. O., et al., Science 253, 323-325 (1991); Sandhu, F. A., et al., J. Biol. Chem. 266, 21331-21334 (1991); Kawabata, S. Nature 354, 476-478 (1991)) have been produced. Results obtained in the different studies appear to depend upon the source of promoter and the protein coding sequence used. For example, Wirak, et al. (1991) found that in transgenic mice expressing a form of the .beta. peptide, intracellular deposits of "amyloid-like" material, reactive with antibodies prepared against APP were observed but did not find other histopathological disease symptoms. The intracellular nature of the antibody-reactive material and the lack of other symptoms suggest that this particular transgenic animal is not a faithful model system for Alzheimer's disease. Kawabata et al. (1991) report the production of amyloid plaques, neurofibrillary tangles, and neuronal cell death in their transgenic animals. In each of these studies, the same peptide fragment, the .beta. peptide plus the 56 remaining C terminal amino acids of APP, was expressed. Wirak et al. (1991) used the human APP promoter while Kawabata, et al. (1991) used the human thy-1 promoter. In transgenic mice expressing the APP751 cDNA from the neuron-specific enolase promoter of Quon, D., et al., Nature 352, 239-241 (1991), extracellular deposits of material reactive with antibody prepared against APP were observed. What was not shown was whether the deposits contained full-length APP751 or .beta. peptide or both, thus precluding any correlation of the deposits with those present in Alzheimer's disease. Quon et al. (1991) also state that the protein encoded by the APP695 cDNA expressed from the neuron-specific enolase promoter, does not form extracellular immunoreactive deposits. These results raise the possibility that although the B peptide is included within the APP695 precursor, use of the neuron-specific enolase promoter in conjunction with the APP695 cDNA may not present an effective Alzheimer's disease model. Furthermore, the presence of APP immunoreactive deposits is not correlated with the age or gene dosage in their particular transgenic model.
Alzheimer's disease is a complex syndrome involving pathological and behavioral aspects. A useful disease model should take these complexities into account. There are multiple proteins expressed from the gene with certain forms predominating in a given tissue. In the brain, the 695 form is predominant, but the mRNAs for additional forms are also present (Golde et al., Neuron 4; 253-267 (1990)). It is not known whether the ratio of the different forms changes with the age of the individual. The various protein forms result from alternative splicing such that the KI domain and/or the OX-2 domain may or may not be present in the mature protein. Moreover, the .beta.-peptide results from post-translational processing of the precursor protein. This process can change in time as an individual ages, and can be affected by mutations not directly affecting the structure of the .beta.-peptide: for example, the familial Alzheimer's disease (FAD) mutations at amino acid position 717 in the full length protein (Groate, et al., 1991; Murrell, et al., 1991; Chartier-Harlin, et al., 1991). Given these considerations, the production of universal animal models for Alzheimer's disease necessitates the construction of animal models that take into account the effects of known mutations on the phenotype resulting from the expression of these forms, and the possibility of the ratio of the different forms changing during the lifetime of the animal.
It is therefore an object of the present invention to provide an animal model for Alzheimer's disease that is constructed using transgenic technology.
It is a further object of the present invention to provide transgenic animals that accurately reflect the expression of different forms of the amyloid precursor protein.
It is a still further object of the present invention to provide transgenic animals characterized by certain genetic abnormalities in the expression of the amyloid precursor protein.